A fast-track to fungal diagnosis: the potential of molecular diagnostics for fungi at the point of care

KEYWORDS:

• Fungal infections
• molecular diagnostics
• point-of-care
• rapid testing
• immunocompromised

1. Global burden and impact of fungal infections

Fungal infections contribute significantly to morbidity and mortality worldwide in patients with both intact and compromised immunity. In the United States alone, among 35.5 million inpatient visits documented in 2018, >666,000 fungal infections were diagnosed with an estimated attributable cost of 6.7 billion dollars [1]. While the early phases of the human immunodeficiency virus (HIV) epidemic drew increased attention to opportunistic infection with fungal pathogens such as Pneumocystis jirovecii and Cryptococcus neoformans, the advent of highly effective antiretroviral therapy and the rise of immunosuppressive therapies for the treatment of chronic organ dysfunction, hematologic malignancies (such as tyrosine kinase inhibitors, which have been associated with invasive aspergillosis) and autoimmune diseases (such as tumor necrosis factor $\mathit{\alpha }$ inhibitors and interleukin antagonists, which have been associated with endemic mycoses) have shifted the epidemiology of invasive fungal infections (IFI) in high-income countries toward an HIV-negative immunosuppressed population [2,3]. In low and middle-income countries, patients living with HIV/AIDS continue to experience the lion’s share of opportunistic fungal infections. IFI, especially involving Aspergillus species, has also been described as an opportunistic phenomenon in the general population following antecedent severe respiratory viral infection such as influenza and, more recently, SARS-CoV-2 infection [4]. The burden of IFI is projected to grow as the number of patients at higher risk increases – a trend that has already been observed in the first two decades of the 21^st century – highlighting the need for rapid and accurate diagnostic testing for IFI [5]. Though associated with lower mortality, superficial fungal infections such as mucocutaneous candidiasis, ringworm, mycetoma, and chromoblastomycosis affect a greater share (20–25%) of the world’s population and lead to an enormous worldwide burden of morbidity that is especially pronounced in developing countries [6].

2. Need for improved fungal diagnostics

Diagnosis of localized, invasive, or disseminated fungal infections remains challenging. Gold-standard mycological diagnostic testing with culture or histopathology allows for species-level identification, antifungal susceptibility testing, and confirmation of tissue-invasive disease but is fraught by limited sensitivity, the need for skilled operators, careful interpretation, and prolonged turnaround times. Adjunctive diagnostics using antibody, antigen, and molecular-based methods have the advantage of increased sensitivity but are limited by high rates of cross-reactivity, long turnaround times due to centralized testing and high cost. Together, these drawbacks substantially reduce the clinical actionability of standard fungal diagnostics, a limitation that is amplified when time and resources are in short supply.

3. POC testing for fungal infections

Defined by the WHO as tests that are affordable, sensitive, specific, user-friendly, rapid/robust, equipment-free, and deliverable to end-users (ASSURED), point-of-care (POC) tests address several limitations of standard fungal diagnostics including cost, complexity, and turnaround time [7]. From clinical, epidemiologic and public health perspectives, the capability to detect fungal pathogens from clinical samples rapidly while maintaining high analytical performance has the potential to transform fungal diagnosis in several ways. First, POC tests can facilitate the diagnosis of fungal infections in low- and middle-income countries where access to centralized and expensive laboratory testing is unavailable. Second, POC testing would allow for a more rapid clinical response to fulminant and rapidly progressive fungal infections or an infection control and containment of fungal infections with outbreak potential and/or those caused by pathogens harboring significant antimicrobial resistance such as Candida auris. Improved antifungal stewardship may be a related additional benefit. Third, POC tests may augment existing outbreak investigation and mitigation strategies through rapid diagnosis and abrogating fungal spread. Fourth, rapid, easy-to-use diagnostics for common superficial fungal infections could be deployed in home-based self-testing strategies or in outpatient (clinic, urgent care, and emergency room) settings, reducing cost and improving allocation of resources across a system of healthcare in resource-limited and higher-resource settings alike.

4. Existing POC tests for fungal infection

Current POC methodologies, which mostly rely on rapid antigen or antibody testing via lateral flow immunoassay (LFIA) or lateral flow assay (LFA), have been effectively applied to fungal diagnosis. Several LFAs have been developed for the diagnosis of invasive aspergillosis. One such test, the Aspergillus lateral flow device (LFD), detects extracellular glycoprotein antigen secreted by Aspergillus fumigatus in human serum within 15 min using a mouse monoclonal antibody and has shown promising results [8]. Some evidence supports that Aspergillus LFD testing is as specific as, and possibly more sensitive than, the standard galactomannan assay based on ELISA [9]. The cryptococcal antigen (CrAg) LFA, an immunochromatographic POC test for the qualitative or semiquantitative detection of Cryptococcus species polysaccharide antigens in serum or cerebrospinal fluid, is inexpensive and yields results in 10 min. Both tests perform similarly to existing non-POC antigen-based comparator testing. CrAg LFA POC testing has been implemented successfully in resource-limited settings for diagnosis of cryptococcal meningitis [10]. LFA-based assays for histoplasmosis and coccidioidomycosis have also been developed [11,12]. However, POC tests for other pathogens including endemic fungi, Pneumocystis, members of the order Mucorales, talaromycosis, sporotrichosis, paracoccidioidomycosis, and other agents superficial fungal infections have been more elusive.

5. Molecular POC tests for fungal infection

In the past two decades, molecular methods have become central to the diagnosis of many infectious diseases due to several advantages over conventional methods including enhanced sensitivity and specificity, and the provision of genotypic virulence or resistance information [13]. However, while molecular methods are widely utilized for bacterial and viral diagnosis, their role in the diagnosis of fungal infections is comparatively limited due to several technical factors including difficulties in lysing mold cell walls for nucleic acid extraction, sample contamination by ubiquitous environmental fungi, uncertainties regarding the optimal clinical specimen type to test, particular primer selection, and difficulties validating a given polymerase chain reaction (PCR) test, all of which limit result standardization [14]. In addition, molecular tests have historically required turnaround times on the order of hours, expensive equipment, and platform user training due to their high complexity, all of which limit POC application. While molecular diagnostics for Aspergillus, Candida, and Pneumocystis have been successfully incorporated into the laboratory setting, deployment at POC has been limited thus far. Advantages of bringing molecular testing to POC, even compared to existing POC antigen-based testing, include enhanced sensitivity, the ability to probe for resistance genes, extended subtyping for epidemiologic purposes, and multiplexed platforms that may be deployed at large scale.

In recent years, a paradigm shift toward molecular POC testing for fungal infections has begun to take shape, prompted by the development of novel technologies that eliminate the need for high-footprint thermal cyclers required for PCR. One such method is loop-mediated isothermal amplification (LAMP), which relies on the use of at least four primers that recognize six separate regions of the target nucleic acid sequence and yields a self-primed deoxyribonucleic acid (DNA) molecule through the use of a polymerase with helicase activity at isothermal temperatures [15]. Based on the potential for low-complexity, low-footprint, low-cost, and rapid diagnosis, LAMP, and other novel technologies such as proximity ligation assay (PLA) have been applied to several investigational or early-development molecular assays for fungal diagnosis with POC applicability. A recent meta-analysis assessing the diagnostic performance of LAMP-based assays for the detection of fungal pathogens in clinical samples supports this potential [16].

LAMP- and PLA-based assays have been developed for the diagnosis of aspergillosis, with promising performance characteristics in preliminary studies [17,18]. LAMP-based methods can also identify drug resistance in Aspergillus species [19]. The rapid and accurate diagnosis of superficial, semi-invasive, and invasive aspergillosis is especially relevant to resource-limited settings where laboratory testing is prohibitively expensive or unavailable, for epidemiologic surveillance of resistance, and to high prevalence settings (such as natural disasters or pandemics) where lower-cost and rapid technologies are needed. A spike in cases of post-COVID aspergillosis and mucormycosis in India associated with delayed diagnosis of high mortality during the early phases of the SARS-CoV-2 pandemic illustrates the unmet need in this arena [20,21]. A LAMP-based assay targeting the Hcp100 locus of H. capsulatum or the internal transcribed spacer region (ITS) has been developed with similar potential for deployment in resource-limited settings at POC where laboratory-based antigen testing is not readily available [22]. Such a technology would be especially useful in highly endemic, resource-limited settings such as Guatemala, where histoplasmosis is the most common AIDS-defining condition [23]. Investigational LAMP-based assays for talaromycosis and paracoccidioidomycosis with reduced turnaround times compared to standard testing could be similarly leveraged in endemic regions of Southeast Asia and South America [24,25]. LAMP-based assays with turnaround times of under 30 min and high sensitivity and specificity have been developed for the detection of Cryptococcus with primers against capsular-associated protein 10 gene (CAP10) and for Pneumocystis using primers to the 18S rRNA gene [26,27]. These assays are most applicable to resource-limited settings with high prevalence of HIV-associated Cryptococcus and Pneumocystis infection such as sub-Saharan Africa. LAMP assays for the detection of Candida species using ITS primer targets have several potential applications at the POC including for rapid and accurate diagnosis of candidiasis (particularly for oral, cutaneous, and vaginal candidiasis) in home and outpatient settings as well as for outbreak investigation and containment [28,29]. LAMP has been used to identify C. auris, a pathogen of particular epidemiologic concern due to a propensity for multidrug resistance and environmental persistence and transmission [30]. LAMP-based technology has been extended to the fungal causes of chromoblastomycosis and mycetoma, which cause significant morbidity in the developing world [31].

6. Conclusions and future directions

Rapid and accurate fungal diagnostic testing remains urgently needed to mitigate the burden of fungal infection worldwide. While some rapid antigen testing is available for certain significant fungal pathogens, bringing molecular testing to the POC is likely to facilitate and accelerate fungal diagnosis and therefore the clinical and public health management of fungal diseases. The combination of high sensitivity, specificity, low operator complexity, and shortened turnaround time of LAMP-based or similar molecular tests promises to address this urgent need. As additional species-specific assays are developed, validation in larger patient cohorts will be necessary to ascertain their performance and confirm their clinical and epidemiologic roles. Broad adoption of accurate and reliable molecular POCs for fungal infections will be contingent on optimal device design with minimal maintenance requirements, decreased cost compared to centralized testing, processes for quality assurance and regulatory oversight, appropriate evidence provision, and stakeholder engagement including patients, clinicians, testing operators, and healthcare management personnel [32,33].

Declaration of financial/other relationships

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

Additional information

Funding

This paper was not funded.